With the rapid growth of research and development in the field of biotechnology there has been an ever increasing demand for new methods and materials critical to the efficient performance of biotechnology research. Indeed, whole new industries have evolved to support the biotechnology revolution. One technology area that has received much attention in service of the needs of the biotech industry is that of chromatographic systems. The separation and purification of biomolecules is of paramount importance, not only to the molecular biologist performing preliminary cloning experiments, but also to the biochemical engineer responsible for the commercial production of high purity products. Much emphasis has been placed on the adaptation of traditional chromatography techniques and systems to meet the many special purification problems of the biotechnology industry. The literature is replete with disclosures of chromatographic theories, techniques and materials for separation of purification of biomolecules.
Types of chromatography which have been applied to the purification of biomolecules, that is, molecules derived from biological sources, include size exclusion chromatography, ion exchange chromatography, bioaffinity chromatography, reversed phase chromatography and hydrophobic interaction chromatography, among others. The application and efficiency of each of those types of chromatography procedures relies on the selectivity of surface-surface interactions between the solute molecules and the stationary phase of the chromatography system, each interacting with the mobile liquid phase. A wide variety of stationary phase chromatography support materials are commercially available.
The present invention is directed to a method for preparation of new stationary phase chromatographic support materials and other surfaces, which are designed to mimic the structure and of biological cell membranes. Consequently, separation of biomolecules in chromatographic systems utilizing the immobilized artificial membrane supports of the present invention are the result of molecular interactions similar to the interactions of said biomolecules and biological membranes in vivo. More specifically, use of the present supports allow separation of a wide variety of peptides/proteins using an aqueous mobile phase without (or with minimum use of) the added protein-denaturing solvents commonly used in the now popular reversed-phase chromatographic systems.
The present compositions prepared in accordance with the invention having covalently bound artificial membrane structure can be employed in chromatographic systems using a highly polar or non polar mobile phase. They also find use as catalytic surfaces for biochemical reactions, coatings for biosensors, surfaces for antigen presentation, and in other applications where membrane mimetic functionality is desired.
The immobilized artificial membrane-bearing chromatographic supports of this invention also find application to a novel generic protein purification procedure for hybrid proteins expressed by genetically engineered microorganisms. A biologically active protein is expressed as a hybrid protein covalently linked to a membrane binding peptide through a selectively cleavable peptide linkage. The hybrid peptide is first purified using an immobilized membrane chromatographic support in accordance with this invention, and thereafter, the hybrid peptide is subjected to predetermined conditions for selective cleavage at a cleavage site between the active protein and the membrane binding peptide.
Possibly more significant than use of the present chromatographic supports as a tool for separation and purification of biomolecules is the potential offered by use of the present supports in a high performance chromatographic system for studying the interaction between solute molecules in the mobile phase and an immobilized membrane constituent stationary phase which can include receptors, enzymes, antibodies and the like. Thus, the chromatographic supports can serve as a powerful tool for the evaluation and study of drug membrane/membrane constituent interactions, and they can find use as an in vitro indicator of potential or probable drug activity. Moreover, the artificial membrane bearing supports can be used for catalysis reactions and chiral syntheses known to take place in biological or artificial membrane (liposome) environments. The supports will also find use for vaccine preparation in that it will allow isolation and purification of membrane binding fractions of viral homogenates.
Enzyme mimetics attempt to mimic the chemical reaction of an enzyme using smaller, less complex chemical systems. The idea of exactly mimicking an enzyme in every physical-chemical detail and biological activity is absurd; one would have to make the entire enzyme.
Similarly, immobilized artificial membranes is a concept to mimic a particular part of the cell membrane environment on a solid support. Exactly mimicking an entire cell membrane environment on a solid support is technically unachievable. However, liposomes are frequently used as an artificial membrane system to study complex cellular events occurring in membranes. Liposomes are easy to prepare and the researcher can define the lipid matrix in this artificial membrane system.
Covalently binding membrane lipids to silica at high molecular surface densities is thus more closely related to liposomes (artificial membranes) in contrast to cell membranes. Hence, the present solid supports are denoted as immobilized artificial membranes, instead of immobilized cell membranes.
Liposomes contain aqueous cores, bilayers, and mobile lipids. In contrast, the prototype immobilized artificial membrane contains a monolayer of bound lipids lacking lateral mobility. Immobilized bilayers were not synthesized initially because of the complexity of creating a hydrophilic surface of phosphocholine headgroups linked to 30 A of hydrocarbon bonded to silica. We anticipate developing immobilized bilayers, but monolayers of lipids have proved effective for chromatography of biomolecules. Depending on application and the mechanism of interaction, a bilayer many not be necessary.
In accordance with one embodiment of the present invention a composition of matter is provided which comprises a mechanically stable particulate support structure dimensioned for use in a chromatographic system, an artificial membrane structure covalently immobilized on the surface of said support material. The membrane structure comprises an amphiphile having a hydrophilic headgroup portion and a hydrophobic portion. The molecules of the amphiphilic compound collectively define the membrane structure on the surface of the support material so that the membrane structure, in the preferred embodiment, has a hydrophobic inner portion and a hydrophilic outer headgroup portion.
"Immobilized" as used to describe the membrane structure on the surface of the present chromatographic supports is to be regarded as relative to the mobile phase. The molecules of the amphiphilic compound are covalently bonded to the surface for "immobilization".
The preferred amphiphilic compounds forming the immobilized membrane structure are those occurring in artificial membranes (liposomes) and biological cell membranes. Phospholipids are most preferred. The immobilized membrane structures can be modified by adsorption of other biological membrane constituents, such as lipids, including phospholipids other than that used as the principal membrane-forming phospholipid, peptides/proteins, saccharides and the like.
Preparation of preferred covalently immobilized membrane chromatographic supports in accordance with this invention can be accomplished utilizing a novel phospholipid carboxylates derived by reaction of C.sub.10 -C.sub.16 cyclic dicarboxylic acid anhydrides with glycero-phosphatides and lysophospholipids.